Summary: Our studies are directed toward understanding 1) the structure and function of human interferon-alphas and their receptors and 2) the mechanisms by which they elicit their pleiotropic biological activities. In an effort to determine the domain(s) of IFN-alpha 21a that are responsible for its distinctive antiproliferative and binding activities, we have employed genetic engineering to construct a number of interferon hybrids using as parents IFN-a21a and IFN-a2c. They exhibit distinct biological and binding properties. The hybrids have a similar antiviral specific activity on bovine cells but differentantiproliferative and binding properties on human Daudi cells. If the 76-166 amino acid residue region is IFN- a2c (76-95)/IFN-a 21a (96-166), antiproliferative activity is highest (HY-3). If the 81-95 amino acid residue region is IFN-a2c, antiproliferative activity is greater than seen when the same region is IFN-a 21a. There are only three amino acids which differ between IFN-a2c and IFN-a21a in the 81-95 region. Construction of 8 mutants in this region by site-directedmutagenesis and cassette mutagenesis has shown that tyrosine at positions 86 and 90 is very important in the antiproliferative activity of IFN-a. Studies have shown that if serine (S) 86 and asparagine (N) 90 are replaced by tyrosine (Y), the antiproliferative activity is increased. Also, if Y86 is replaced by isoleucine (I) the antiproliferative activity is comparable. If Y86 is replaced by aspartic acid (D), lysine (K) or alanine (A), however, antiproliferative activity is substantially decreased. This indicates that Y and/or I at position 90 are very important in the antiproliferative activity of IFN-a. Sceondary structure was not changed by replacement at position 86 (as shown by circular dichroism analysis). Thus, changes in biological activities do not appear to be the result of conformational changes. Data from competitive binding assays show that the N-terminal region of the IFN molecule may be important in the binding activities of IFN. If the N-terminal region of the IFN has the IFN-a2c sequence it competes well with 125I-IFN-a2b. Each of the hybrids and mutants which compete well with 125I-IFN-a21a have the IFN-a21a sequence at their N-termini and have either tyrosine or isoleucine at positions 86 or 90. The signal transduction properties of HY-2 [IFN- a 21a (1-95)/a2c (96-165)] and HY-3 [IFN-a 2c (1-95)/a21a(96-166)] were evaluated by Electrophoretic Mobility Shift Analysis (EMSA) and RNase ProtectionAssays. Both HY-2 and HY-3 induced activation of STAT 1 and 2. Additionally, at concentrations where no AP activity was seen, HY-2 induced avariety of IFN responsive genes to the same degree as HY-3. RNase Protection Assays also indicate that, at concentrations where no AP activity was seen for HY-2, this construct retained the ability to induce a variety of IFN-inducible genes. These data suggest that the antiproliferative response may not be solely directed by the STAT 1 and 2 pathways in a non-hematopoietic and a hematopoietic cell line and in primary human lymphocytes. The above hybrid work was filed as a patent application (NIH Reference# E-068-98/1) on June 29, 1999. We have compared the antigenic properties of human IFN-a2c, IFN-a21a, IFN-a21a/a2c hybrids and their mutants using a panel of 26 anti-IFN monoclonal antibodies (anti-IFN-a1, a2 and a 8/1/8) and have found IFN-a2c and IFN-a21a to be antigenically distinct. Analysis of antigenic properties of hybrid polypeptides consisting of IFN-a21a at theN-terminal (1-75) and IFN-a2c at theC-terminal (76-166) with monoclonal antibodies recognizing the respective region of IFN-a2c showed antigenic distinction among the constructs tested. In addition, circular dichroism (CD) spectra analysis of mutants displaying the weakest reactions with the monoclonal antibodies used showed insignificant differences in secondary structure between the constructs and IFN-a2c. Lysates from cells grown in the presence of our interferon constructs are being analyzed by 2D electrophoresis and tested for reaction to various antibodies directed against proteins of different signaling pathways (e.g. Cytokine signaling pathway, MAP Kinase pathway, JAK/STAT pathway). Also, total mRNA was purified from cells treated with our constructs and assayed by microarray against human cDNA. In addition, we are using the IFN-alpha hybrids in collaborative studies with CBER scientists to examine their inhibition of HIV1 replication in primary monocytes and T cells. We are also collaborating with the NCI/FDA Clinical Proteomics Program.